Manufacture of fatty acids having straight and long carbon chains using a microorganism

ABSTRACT

A method for the oxidation of hydrocarbons to monocarboxylic acids and then to dicarboxylic acids by the aerobic cultivation or resting cell reaction of the organism Debaryomyces vanriji (BR-308) ATCC 20588.

BACKGROUND OF THE INVENTION

This invention concerns the production of dicarboxylic acids ordicarboxylic acids along with monocarboxylic acids corresponding tohydrocarbons having straight and long carbon chains and/ormonocarboxylic acids having straight and long carbon chains as substrateusing a microorganism.

Monocarboxylic acids having straight and long carbon chains are usefulraw materials of surfactants, detergents, stabilizers, and the like.However, their use has been limited since natural fats, such as beef fatand palm oil, have been mostly employed for the preparation of the abovementioned chemicals.

Dicarboxylic acids having straight and long carbon chains are useful rawmaterials for the preparation of plasticizers, synthetic resins,synthetic lubricants, oils, perfumes, and the like. The establishment ofa method of the manufacture of dicarboxylic acids with varied carbonnumbers on an industrial scale from petroleum derived feedstocks hasbeen desired.

Microbial production of monocarboxylic acids and dicarboxylic acids iswell known. In these reported reactions, normal paraffins contained inpetroleum distillate are used as substrate for corresponding mono- anddi-carboxylic acids. Further, the use of natural as well as syntheticmonocarboxylic acids as raw materials for microbial conversion todicarboxylic acids has been reported: VanderLinden and Thijsse, "TheMechanisms of Microbial Oxidations of Petroleum Hydrocarbons"; Advancesin Enzymology, Vol. 27, p. 469 (1965); Y. Minura, U.S. Pat. No.3,793,153; S. Akabori, et al., U.S. Pat. No. 3,843,466.

Commercially advantageous methods have not yet been established.

SUMMARY OF THE INVENTION

It has now been found that a yeast strain which belongs to Debaryomycesgenus can produce dicarboxylic acids or dicarboxylic acids along withmonocarboxylic acids by oxidizing hydrocarbons or mixtures ofhydrocarbons and moncarboxylic acids having straight and long carbonchains.

As described herein, this invention is characterized by using a yeaststrain belonging to the Debaryomyces genus, Debaryomyces vanriji; inorder to produce dicarboxylic acids or dicarboxylic acids along withmonocarboxylic acids advantageously.

Utilizing this microorganism, this invention produces, (1) dicarboxylicacids or a mixture of dicarboxylic acids and monocarboxylic acidscorresponding to the hydrocarbons having straight and long carbon chainswhich have been used as the substrate, (2) dicarboxylic acidscorresponding to natural as well as synthetic monocarboxylic acids usedas raw material and (3) dicarboxylic acids corresponding to a mixture ofhydrocarbons having long, straight carbon chains and monocarboxylicacids of similar chain length when used as substrate.

DETAILED DESCRIPTION OF THE INVENTION

The carboxylic acid producing microorganism, Debaryomycesvanriji(BR-308), employed in this invention was collected from the soilnear a petroleum refinery in Akita Prefecture and was isolated for use.Said microorganism was identified as Debaryomyces vanriji from thefollowing micrological properties. The strain has been deposited withthe American Type Culture Collection with the accession number ATCC20588.

The properties of said microorganism are described below:

1. Shape and size:

Growth in malt extract: After 3 days at 25° C. the cells are sphericalto oval, (3-8)×(4-20)u; single or in groups. A sediment and a thin,dull, creeping pellicle are formed.

Growth on malt agar: After 7 days at 25° C. the colony is whitish ordark yellowish, dull to shiny, smooth with slightly sinuous margin.

Slide Culture on potato--and corn meal agar: A primitive pseudomyceliumis abundantly formed.

2. Formation of ascospores:

Ascospores are formed on 1/8 M Van't Hoff's gypsum blocks and V 8 agar.The spore are spherical or oval.

3. Fermentation of sugars: Negative.

4. Assimilation of carbon compounds: See Table 1.

5. Splitting of arbutin: Positive.

6. Assimilation of KNO₃ : Negative.

7. Growth in vitamin-free medium: Positive.

8. Growth at 37° C.: Positive.

                  TABLE 1                                                         ______________________________________                                        Assimilation of carbon compounds                                              ______________________________________                                               Glucose          +                                                            Galactose        +                                                            L-sorbose        +                                                            Sucrose          +                                                            Maltose          +                                                            Cellobiose       +                                                            Trehalose        +                                                            Lactose          -                                                            Melibiose        +                                                            Soluble Starch   +                                                            D-xylose         +                                                            Ethanol          +                                                            Glycerol         +                                                            Salicin          +                                                            Inositol         -                                                     ______________________________________                                    

In this invention, hydrocarbons having 10 to 18 carbon atoms areappropriate raw materials (substrate) for the production of dicarboxylicacids or a mixture of di- and monocarboxylic acids especiallyhydrocarbons of 11 to 16 carbon chain length are desirable. For theselective production of dicarboxylic acids, monocarboxylic acids andhydrocarbons having a skeletal length of 10 to 18 carbon atoms each aresuitable precursors, hydrocarbon skeletal lengths of 11 to 16 carbonnumbers are preferred.

The oxidation reaction of this invention is a typical resting cellphenomenon and can be carried out in an aqueous buffer solution, as forexample a phosphate buffer solution of pH 7.

The reaction can also be carried out in a growth medium which containsnutrient for the yeast. The reaction then becomes acultivation-oxidation reaction. In this mode of operation, the mediashall contain the usual nutrient material including an assimilablecarbon source, nitrogen source, and appropriate vitamins and minerals,all well-known to those skilled in the art.

Carbon sources appropriate to a growth medium can include for exampleglucose, sucrose, maltose, the substrate hydrocarbon or monocarboxylicacid, and the like.

Nitrogen sources can include inorganic nitrogen compounds as for exampleammonium nitrate, ammonium phosphate, or the like; and organicnitrogen-containing materials as for example peptone, corn steep liquor,and amino acids.

Vitamins and minerals needed for growth can include sodium phosphate,calcium phosphate, magnesium sulfate, zinc sulfate, ferrous sulfate,manganese sulfate as minerals and yeast extract and the like asvitamin-containing additive.

When the oxidation is carried out as a resting cell reaction, it becomesnecessary to grow up a healthy cell mass prior to oxidation. This cellmass is best prepared by growing the yeast culture in the above growthmedium prior to oxidation. The whole cell culture including cells andnutrient material can be used in the oxidation reaction, or the cellmass can be removed from the spent nutrient by centrifugation orfiltration prior to adding the cell mass to the substrate.

Thus, in this invention, the strain of carboxylic acid producingmicroorganisms, Debaryomyces vanriji(BR-308) ATCC 20588, or a culturethereof or cells of the strain cultured previously are added to themedium containing the substrate to carry out the reaction and agitated,aerated through a nozzle, or shaken so that the microorganism cancontact the components of the medium thoroughly.

The reaction temperature is kept at 25° to 35° C. and pH is controlledat 3 to 9, preferably pH 4 to 8. The period of time of reaction dependson the substrate to be used, but usually a reaction takes 24 to 120hours to finish completely.

We have found it advantageous to use a mixed substrate containing atleast some monocarboxylic acid for the reason that the monocarboxylicacid tends to increase solubility of the substrate in the aqueousreaction mixture and to suppress accumulation of additionalmonocarboxylic acid during oxidation.

A readily available source of the monocarboxylic acid for this mixedsubstrate is from the accumulation of the acid from previous reactions.

When cultivation (reaction) is carried out as described above, thesubstantial amount of dicarboxylic acids or a mixture of dicarboxylicacids containing monocarboxylic acids is produced and accumulated. Thesecarboxylic acids are separated and purified by a conventional methodsuch as extraction, solid-liquid separation, neutralization-extractionand fractional distillation, and then harvested as monocarboxylic acidsand dicarboxylic acids, or a mixture of both.

As illustrated by the preferred embodiment described below, the organismof this invention produces monocarboxylic acids and dicarboxylic acidsin high yields by well known cultivation methods using the newmicroorganism. Therefore, it is believed that this invention contributesgreatly to the production or carboxylic acids with straight and longcarbon chains.

EXAMPLE 1

Medium composition for flask cultivation

Sucrose: 30 g

NH₄ Cl: 4 g

KH₂ PO₄ : 2 g

MgSO₄.7H₂ O: 0.6 g

ZnSO₄.7H₂ O: 0.01 g

FeSO₄.7H₂ O: 0.01 g

Mycological peptone: 0.5 g

Yeast extract: 0.5 g

The components for pre-cultivation were dissolved in distilled water tomake the total volume 1 liter and adjusted to pH 6.5. One hundredmilliliters of the resulting media was added to a 500 ml flask andsterilized in an autoclave at 121° C. for 15 minutes. Cells ofDebaryomyces vanriji(BR-308) ATCC 20588 which had been grown on maltextract agar at 30° C. for one month were inoculated (3 loopsful) to theabove sterile medium and were cultured on a reciprocal shaker at 30° C.for 29 hours.

Medium composition for fermentation

KH₂ PO₄ : 10 g

NH₄ Cl: 5 g

MgSO₄.7H₂ O: 0.6 g

FeSO₄.7H₂ O: 0.01 g

ZnSO₄.7H₂ O: 0.008 g

Mycological peptone: 0.5 g

Yeast Extract: 0.5 g

The above-enumerated components for a fermentation medium were dissolvedinto distilled water to make the total volume 1 liter and sterilized at121° C. for 15 minutes; 800 ml of the medium and 110 g of theappropriate hydrocarbons or carboxylic acids as shown in Table 2 weresterilized in an autoclave at 115° C. for 15 minutes.

Into a 2 l fermentor were placed 100 ml of the precultivation medium,800 ml of the fermentation medium and 110 g of sterilized reactantsubstrate. The mixture was allowed to reach with aeration and agitationat 30° C. for 96 to 120 hours at pH 6.5 to pH 7.5. 2 N potassiumhydroxide was used for pH adjustment. When foaming was observed duringthe cultivation, small quantities of a solution of 15% defoaming agent(manufactured by Toshiba Silicone Co.; TSA 730) which had previouslybeen autoclaved at 115° C. for 15 minutes were poured into the culturemedium. On finishing cultivation, solid potassium hydroxide was added tothe culture broth to pH 10, the broth emptied from the fermentor,filtered under the reduced pressure using filter aid, and washed.Products were extracted by ether, methylated with diazomethane after theether was removed and analyzed by gas chromatography.

The results are shown in Table 2.

                  TABLE 2                                                         ______________________________________                                                 Concentration of acids in the medium after                                    96- 120 hours cultivation (mg/L)                                                Monocarboxylic                                                     Substrate  Acids        Dicarboxylic Acids                                    ______________________________________                                        n-Decane   Capric           1,8-Octane                                                   Acid       6.0   DCA        4.1                                    n-Undecane Undecyl          1,9-Nonane                                                   Acid      10.7   DCA        9.1                                    n-Dodecane Lauric           1,10-Decane                                                  Acid      11.3   DCA        16.0                                   n-Tridecane                                                                              Tridecyl         1,11-Undecane                                                Acid      19.1   DCA        13.9                                   n-Tetradecane                                                                            Myristic         1,12-Dodecane                                                Acid      24.5   DCA        12.8                                   n-Pentadodecane                                                                          Pentadecyl       1,13-Tridecane                                               Acid      18.4   DCA        14.8                                   n-Hexadecane                                                                             Palmitic         1,14-Tetradecane                                             Acid      19.1   DCA        18.3                                   Pelargonic Acid             1,7-Pentan DCA                                                                           6.3                                    (C.sub.8 H.sub.17 COOH)                                                       Lauric Acid                 1,10-Decane DCA                                                                          5.3                                    (C.sub.11 H.sub.23 COOH)                                                      Palmitic Acid               1,14-Tetradecane                                  (C.sub.15 H.sub.31 COOH)    DCA        17.3                                   A mixture of                1,10-Decane DCA                                                                          18.0                                   n-Tridecane and                                                               Lauric Acid con-                                                              taining equal wt.                                                             ______________________________________                                    

EXAMPLE 2

Components for the fermentation medium described in Example 1 as well as50 g sucrose were dissolved in 1 liter distilled water and adjusted topH 5.5. Fifty milliliters of the medium were placed in a 500 ml shakingflask and inoculated with 2 loopsful of the Debaryomyces vanriji strainused in Example 1, and growth was maintained at 30° C. for 26 hours. Theobtained culture broth was centrifuged in order to separate the cells(about 1 g dry weight). A reaction solution was prepared by mixing 100ml of 0.5 M phosphate buffer (pH 7.0) with 10 ml of normal dodecane. Areaction was carried out between this reaction solution and the filteredcells at 30° C. for 72 hours. The reaction products were basified to pH10 with potassium hydroxide and were analyzed using the same process asin Example 1. As a result, 4.8 mg/L of lauric acid and 3.7 mg/L of1,10-decane dicarboxylic acids were produced in the reaction medium. Forthis experiment, the medium, substrate, and the equipment wereautoclaved previously so that the experiment could be carried out understerile conditions.

We claim:
 1. In the production of mono- and di-carboxylic acids of thesame carbon skeletal length from straight chain C₁₀ -C₁₈ hydrocarbons byaerobically cultivating the hydrocarbons with a microorganism in anutrient medium, at 25°-35° C. and pH 3-9 for 24 to 120 hours, theimprovement which comprises using as the microorganism Debaryomycesvanriji (BR-308), ATCC
 20588. 2. In the production of C₁₀ -C₁₈dicarboxylic acids of the same skeletal length from monocarboxylic acidsby aerobically cultivating the monocarboxylic acid with a microorganismin a nutrient medium, at 25°-35° C. and pH 3-9 for 24 to 120 hours, theimprovement which comprises using the microorganism Debaryomycesvanriji, (BR-308), ATCC
 20588. 3. In the production of dicarboxylicacids of the same carbon skeletal length from straight chain C₁₀ -C₁₈hydrocarbons and monocarboxylic acids by aerobically cultivating thehydrocarbon and the monocarboxylic acid with a microorganism in anutrient medium, at 25°-35° C. and pH 3-9 for 24 to 120 hours, theimprovement which comprises using at the microorganism Debaryomycesvanriji, (BR-308), ATCC
 20588. 4. In the production of mono- anddi-carboxylic acids of the same carbon skeletal length from straightchain C₁₀ -C₁₈ hydrocarbons by growing a microorganism in a nutrientmedium, at 25°-35° C. and pH 3-9 for 24 to 120 hours, separating thecell mass comprising the microorganism and allowing the cell mass toreact aerobically with the hydrocarbon, the improvement which comprisesusing the microorganism Debaryomyces vanriji, (BR-308), ATCC 20588.